Interleukin-12 as an adjuvant for paramyxoviridae vaccines

ABSTRACT

A method is disclosed of reducing viral replication of a virus of the paramyxoviridae family in a host, comprising administering to the host an antigen of the virus in combination with an effective adjuvant amount of interleukin-12 (IL-12). Human viruses of the paramyxoviridae family include paramyxoviruses (e.g., parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3 and parainfluenza virus 4), morbilliviruses (e.g., measles virus) and pneumoviruses (e.g., respiratory syncytial virus); other non-human viruses of the paramyxoviridae family include canine distemper virus, bovine respiratory syncytial virus, Newcastle disease virus and rhinderpest virus. A composition is also disclosed comprising a mixture of an antigen of a virus of the  Paramyxoviridae  family and an effective adjuvant amount of interleukin-12 (IL-12).

RELATED APPLICATION(S)

This application is a Continuation of U.S. application Ser. No.08/980,160 filed on Nov. 26, 1997, which is a Continuation of U.S.application Ser. No. 08/318,480 filed on Oct. 5, 1994. The entireteachings of U.S. application Ser. No. 08/980,160 and U.S. applicationSer. No. 08/318,480 are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grantROA-AI-33933 from the National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV), a member of the Pneumovirus genus ofthe Paramyxoviridae family, is an important cause of respiratory diseasein infants and children (Connors, M., et al., J. of Virol., 66:7444-7451(1992). The immunological basis for the differing susceptibility amongindividuals, and for the limited age range at which severe illnessoccurs, remains unclear.

The major impediment to advancing new candidate vaccines directedagainst RSV to clinical trials is an incomplete understanding of thevaccine-enhanced illness caused by formalin-inactivated RSV vaccines inthe 1960's. Clinical trials of a formalin-activated alum-precipitatedRSV vaccine in the 1960's showed that the vaccine elicitedcomplement-binding antibodies but failed to protect against infection inchildren. In addition, the illness after subsequent infection wasunusually severe with some deaths, and an increased rate ofhospitalization (Kapikian, A. Z., et al. Amer. J. Epidem., 89:405(1969); Fulginti, V. A., et al, Amer. J. Epidem., 89:435 (1969); Kim, W.H., Amer. J. Epidemol., 89:422 (1969); Chin, J. R., et al. Amer. J.Epidem., 89:449 (1969)). A similar enhanced illness can be induced inmice previously immunized with the formalin-inactivated vaccine upon RSVinfection, but not in mice immunized with live RSV (Conners, M., et al.,J. Virol. 66:7441 (1992); Graham, B. S., et al., Immunol. 151:2032(1993); Alwan, W. H., et al., J. Exp. Med. 179:81 (1994)). Clinicaltrials of live attenuated RSV vaccine products have not been associatedwith enhanced illness. Although the live RSV vaccines did not result inenhanced pulmonary disease upon natural infection, the vaccines were, inother respects, as equally unsuccessful as the formalin-inactivatedalum-precipitated RSV vaccines (Kim, W. H., et al., Pediatrics, 48:745(1971); Kim, W. H., et al., Pediatrics, 52:56 (1973); Belshe, R. B., etal., J. Infect. Dis., 145:311 (1982); Wright, R. B., et al., Infect.Immun., 37:397 (1982). Temperature-sensitive mutants of RSV,cold-adapted RSV or live RSV given parenterally have been consideredunsuccessful as vaccines because of high rates of reversion towild-type, unacceptable virulence or lack of immunogenicity in theappropriate age group (Graham, B. S., et al., J. of Immun.,151:2032-2040 (1993).

Thus, a need exists for development of efficacious methods ofvaccination against RSV and for vaccine compositions.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that IL-12 has a potentadjuvant effect for immunizing against Paramyxoviridae virus infection.In one embodiment, the invention comprises a method of reducing viralreplication of a virus of the paramyxoviridae family in a host (e.g.,mammalian, including human, and avian) comprising administering to thehost an antigen of the virus in combination with an effective adjuvantamount of interleukin-12 (IL-12). Human viruses of the Paramyxoviridaefamily include paramyxoviruses (e.g., parainfluenza virus 1,parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4 andmumps virus), morbilliviruses (e.g., measles virus) and pneumoviruses(e.g., respiratory syncytial virus); other non-human viruses of theParamyxoviridae family include canine distemper virus, bovine RSV,Newcastle disease virus and rhinderpest virus. In one embodiment, theinvention relates to a method of reducing replication of the respiratorysyncytial virus (RSV) in a host comprising administering to the host anantigen of RSV in combination with an effective adjuvant amount ofIL-12. Thus, the present invention also relates to a method of elicitingan immune response against viruses of the Paramyxoviridae family in ahost, comprising administering to the host an antigen of a virus of theParamyxoviridae family in combination with an effective adjuvant amountof IL-12. The present invention also relates to a method of immunizing ahost against RSV comprising administering to the host a mixturecomprising an antigen of respiratory syncytial virus in combination withan effective adjuvant amount of interleukin-12.

In addition, the present invention relates to a composition comprising amixture of an antigen of a virus of the Paramyxoviridae family and aneffective adjuvant amount of interleukin-12 (IL-12). In one embodiment,the invention relates to a composition comprising an antigen of therespiratory syncytial virus and IL-12.

As shown herein, exogenous. IL-12 treatment administered at the time ofimmunization with RSV antigen, diminishes RSV replication and increasesendogenous IL-12 mRNA expression at the time of subsequent RSVchallenge. This results in a shift from a Th2 to a Th1-like pattern ofcytokine expression and a consequent shift in antibody isotypeutilization. These results demonstrate that IL-12 is a potent adjuvantfor Paramyxoviridae vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of IL-12 treatment versus log 10 plaque formingunits (pfu)/gram lung from the plaque assay illustrating that IL-12administered at the time of immunization has a marked effect on thereduction of viral replication.

FIG. 2 is a bar graph of IL-12 treatment versus averagedα-tubulin-normalized density from the mRNA Northern blots illustratingthat IL-12 enhanced Th1 cell differentiation and produced a shift from aTh2 to a Th1-like response in mice immunized with inactivated RSVimmunogen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of reducing viral replicationof a virus of the Paramyxoviridae family in a host (e.g., mammal,includimg human, avian) comprising, administering to the host, a mixtureof an antigen of the virus and an effective adjuvant amount ofinterleukin-12 (IL-12). Although the method of the present invention isexemplified using RSV, the method can be used to reduce viralreplication of a variety of viruses from the Paramyxoviridae familywhich include human paramyxoviridae viruses, such as paramyxoviruses(e.g., parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus3, parainfluenza virus 4 and mumps virus), morbilliviruses (e.g.,measles virus) and pneumoviruses (e.g., respiratory syncytial virus).Other non-human viruses of the Paramyxoviridae family include caninedistemper virus, bovine RSV, Newcastle disease virus and rhinderpestvirus. In one embodiment, the method of the present invention is used toreduce viral replication of respiratory syncytial virus (RSV) in a host,and comprises administering to the host an RSV antigen and an effectiveadjuvant amount of IL-12.

In another embodiment, the method of the present invention is used toelicit an immune response against a virus of the Paramyxoviridae familyin a host, comprising administering to the host an antigen of the virusand an effective adjuvant amount of IL-12. In addition, the presentinvention relates to a method of immunizing a host against RSVcomprising administering to the host a mixture comprising an RSV antigenand an effective adjuvant amount of IL-12.

An antigen of a virus of the Paramyxoviridae family includes use of thewhole virus (e.g., inactivated or live, attenuated whole virus), anantigenic portion of the virus, and recombinantly produced virus orportions thereof or fusion proteins. In addition, antigens of thepresent invention include nucleic acid sequences which encode an antigenof a virus of the Paramyxoviridae family. Antigenic portions of theviruses of the Paramyxoviridae family include the fusion glycoprotein (Fprotein) and the hemagglutinin-neuraminidase of the parainfluenzaviruses 1, 2, 3, 4; the F protein and the hemagglutinin-neuraminidase ofthe mumps virus; the F protein and the hemagglutinin-neuraminidase ofthe measles virus; and the F protein and the G glycoprotein of the RSV.Other antigenic portions of the Paramyxoviridae family of viruses whichcan be used in the methods and compositions of the present invention,can be determined by those of ordinary skill in the art.

The IL-12 of the present invention can be obtained from a suitablesource for use in the present method. For example, IL-12 can be purifiedfrom natural sources (e.g., human, animal), produced by chemicalsynthesis or produced by recombinant DNA techniques as described inExample 1. In addition, the IL-12 of the present invention includenucleic acid sequences encoding IL-12, as well as the RNAs encoded bysuch nucleic acid sequences. As used herein, “interleukin-12” and“IL-12” refer to interleukin 12, its individual subunits, fragmentsthereof which exhibit IL-12 adjuvant activity and functional equivalentsof “interleukin-12” and “IL-2”. Functional equivalents of“interleukin-12” and “IL-12” include modified IL-12 protein such thatthe resulting IL-12 product has the same adjuvant activity as the IL-12described herein, and nucleic acid sequences which through thedegeneracy of the genetic code encode the same peptide gene product asIL-12 and having the IL-12 adjuvant activity described herein. Forexample, a functional equivalent of “interleukin-12” and “IL-12” cancontain a “silent” codon or amino acid substitution (e.g., substitutionof one acidic amino acid for another acidic amino acid; or substitutionof one codon encoding a hydrophobic amino acid to another codon encodinga hydrophobic amino acid).

IL-12, a heterodimeric cytokine predominantly excreted by the macrophagecells, has been reported to enhance NK cell and CTL activity, tostimulate the differentiation of Th1 cells, and to induce production ofcytokines, such as IFN-γ (Gately, M. K., et al, Cell. Immunol. 143:127(1992); Naume, B., et al, J. Immunol. 148:2429 (1992); Hsieh, C. S., etal Science 260:547 (1993); Manetti, R., et al J. Exp. Med. 177:1199(1993); Chan, S. H., etal, J. Exp. Med. 173:869 (1991); D'Andrea, A., etal, J. Exp. Med. 176:1387 (1992); Macatonia, S. E., et al, Int. Immunol.5:1119 (1993); Tripp, C. S., et al, Proc. Natl. Acad. Sci. USA 90:3725(1993)). IL-12 formerly referred to as natural killer cell stimulatoryfactor or cytotoxic lymphocyte maturation factor functions to activateand to link the innate and acquired immune responses (Kobayashi, M., etal J. Exp. Med. 170:827 (1989); Stern, A. S., et al Proc. Natl. Acad.Sci. USA 87:6808 (1990); Locksley, R. M., et al Proc. Natl. Acad. Sci.USA 90:5879 (1993)). IL-12 promotes differentiation of uncommitted Thelper cells towards the Type 1 (Th1) phenotype (Hsieh, C. S., et alScience 260:547 (1993); Manetti, R., et al J. Exp. Med. 177:1199(1993)). This results in a characteristic constellation of cytokines,such as IFN-γ, and generally promotes cell-mediated immunity (Chan,S.H., et al, J. Exp. Med. 173:869 (1991); D'Andrea, A., et al, J. Exp.Med. 176:1387 (1992) Macatonia, S. E., et al, Int. Immunol. 5:1119(1993); Gately, M. K., et al, Cell. Immunol. 143:127 (1992) Naume, B.,et al, J. Immunol. 148:2429 (1992)). IL-12 has been demonstrated toenhance the immune response and to improve protective immunity inseveral infectious disease models, including Listeriosis, Leishmaniasis,Toxoplasmosis and lymphocytic choriomeningitis virus infection (Tripp,C. S., et al, Proc. Natl. Acad. Sci. USA 90:3725 (1993); Heinzel, F. P.,et al J. Exp. Med. 177:1505 (1993); Sypek, J. P., et al J. Exp. Med.177:1797 (1993); Afonso, L. C., et al, Science 263:235 (1994);Gazzinelli, R. T., et al, Proc. Natl. Acad. Sci. USA 90:6115 (1993);Khan, I. A., et al Infect. Immun. 62:1639 (1994); Orange, J. S. et al, JImmunol. 152:1253 (1994)). The purification and cloning of IL-12 aredisclosed in PCT publication nos. WO 92/05256 and WO 90/05147, and inEuropean patent publication no. 322,827 (identified as “CLMF”).

Interleukin-12 or IL-12 is a mammalian cytokine which exhibits numerousimmunologic effects, including modulation of T cell response to antigens(see, for example, PCT publication nos. WO 92/05256 and WO 90/05147,wherein IL-12 is identified as “NKSF”). It has also been suggestedgenerally that IL-12 might have some application as a vaccine adjuvant(Scott, P., Science, 260:496-497(1993); Trichieri, G., Immunology Today,14:335-338(1993)).

In the method of the present invention, an effective adjuvant amount ofIL-12 is administered in combination with an antigen of a virus of theParamyxoviridae family. That is, the IL-12 is administered at a timeclosely related to immunization of the host with the viral antigen, sothat an enhanced immune response in the host is produced relative to theimmunization of a host in which IL-12 is not administered. Thus, theIL-12 can be administered prior to, preferably just prior to,immunization, at the time of immunization (i.e., simultaneously) orafter immunization (i.e. subsequently). In addition, the IL-12 can beadministered prior to immunization with the viral antigen of theParamyxoviridae family, followed by subsequent injections of IL-12 afterimmunization with the antigen.

The IL-12 and the antigen can be administered to a host in a variety ofways. The routes of administration include intradermal, transdermal(e.g., slow release polymers), intramuscular, intraperitoneal,intravenous, subcutaneous, oral, epidural and intranasal routes. Anyother convenient route of administration can be used, for example,infusion or bolus injection, or absorption through epithelial ormucocutaneous linings. In addition, the IL-12 and the antigen of theParamyxoviridae virus can be administered together with other componentsor biologically active agents, such as other known adjuvants (e.g.,alum, MPL, QS21), pharmaceutically acceptable surfactants (e.g.,glycerides), excipients (e.g., lactose), carriers, diluents andvehicles. If desired, certain sweetening, flavoring and/or coloringagents can also be added.

The IL-12 and the antigen can be administered as a prophylactic vaccineto hosts which are either infected or uninfected with the virus. TheIL-12 and the antigen can also be administered as a therapeutic vaccineto infected hosts and can result in amelioration or elimination of thedisease state caused by the infecting virus.

Further, the antigen and/or IL-12 can be administered by in vivoexpression of polynucleotides encoding such into a mammalian subject.For example, the IL-12 or the Paramyxoviridae antigen can beadministered to a host using live vectors, wherein the live vectorscontaining IL-12 and/or antigen nucleic acid sequences are administeredunder conditions in which the antigen and/or IL-12 are expressed invivo. For example, a host can be injected with a vector which encodesand expresses an antigen of a virus of the Paramyxoviridae family invivo in combination with IL-12 protein or peptide, or in combinationwith a vector which encodes and expresses the IL-12 protein in vivo.Alternatively, a host can be injected with a vector which encodes andexpresses IL-12 in vivo in combination with a Paramyxoviridae antigen inpeptide or protein form, or in combination with a vector which encodesand expresses a Paramyxoviridae antigen in vivo. A single vectorcontaining the sequences encoding a Paramyxoviridae antigen and theIL-12 protein are also useful in the methods and compositions of thepresent invention.

Several expression vector systems are available commercially or can bereproduced according to recombinant DNA and cell culture techniques. Forexample, vector systems such as the yeast or vaccinia virus expressionsystems, or virus vectors can be used in the methods and compositions ofthe present invention (Kaufman, R. J., A J. of Meth. in Cell and Molec.Biol., 2:221-236 (1990)). Other techniques using naked plasmids or DNA,and cloned genes encapsidated in targeted liposomes or in erythrocytesghosts, can be used to introduce the IL-12 and/or Paramyxoviridaeantigen polynucleotides into the host (Freidman, T., Science,244:1275-1281 (199); Rabinovich, N. R., et al., Science, 265:1401-1404(1994)). The construction of expression vectors and the transfer ofvectors and nucleic acids into various host cells can be accomplishedusing genetic engineering techniques, as described in manuals likeMolecular Cloning and Current Protocols in Molecular Biology, which arehereby incorporated by reference, or by using commercially availablekits (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Press,1989; Ausubel, F. M., et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Interscience, 1989).

The amount of antigen used in the methods and compositions of thepresent invention is an amount which produces an effectiveimmunostimulatory response in the host. An effective adjuvant amount ofIL-12 is an amount such that when administered, it results in anenhanced immune response relative to the immune response when aneffective adjuvant amount of IL-12 is not administered. In addition, theamount of antigen from a virus of the Paramyxoviridae family and IL-12used to immunize the host will vary depending on a variety of factors,including the antigen employed, the size, age, body weight, generalhealth, sex and diet of the host, and the time of administration,duration or particular qualities of the Paramyxoviridae virus beingvaccinated against. Adjustment and manipulation of established dosageranges are well within the ability of those skilled in the art.

The formulation and route of delivery of vaccine products can influencethe induction of T helper lymphocyte subsets and may thereby affectdisease expression after viral challenge (Graham, B. S., et al, Immunol.151:2032 (1993)). Depletion of IL-4 at the time of immunization byneutralizing monoclonal antibody induces a Th2 to Th1-like immuneresponse shift, accompanied by an improved clinical outcome and anincreased CD8+ cytotoxic T lymphocyte (CTL) activity (Tang, Y.-W., etal, J. Clin. Invest. (1994)). This was associated with an increasedexpression of endogenous IL-12 message at the time of challenge in theanti-IL-4 treated mice. These findings suggest that selective activationof the Th2-like cell subset may be responsible for RSV vaccine inducedimmunopotentiation of disease and that IL-12 may be associated withshifting the response away from Th2 to a more Th1-like response.

Replication of RSV is markedly reduced after live RSV challenge in micegiven IL-12. Use of IL-12 as an adjuvant in a composition comprised ofan antigen from RSV and IL-12 also induced a shift from a Th2 to aTh1-like immune response in mice after RSV challenge. While the IL-12adjuvant effect was potent for reduction of RSV replication, it is moreimportant for use as a vaccine adjuvant to decrease illness followingchallenge by RSV. The use of cytokines as adjuvants can allow one tocontrol the immune parameters induced by immunization to improveprotective effects and decrease the negative effects of a vaccine forRSV. The effects of IL-12 on the immune responses to RSV vaccination asmeasured after live virus challenge in the BALB/c mouse model aredescribed in the Examples. The results indicate that IL-12 acts as apotent adjuvant and is a useful product to include in RSV vaccines.

As described in Example 1, BALB/c mice were immunized with inactivatedwhole virus intramuscularly, and murine recombinant IL-12 wasadministered intraperitoneally for 5 successive days starting at one daybefore immunization or challenge. The mice were challenged with livevirus 4 weeks later. The viral replication in lungs 4 days afterchallenge was assessed. IL-12 administered at the time of immunizationhad a marked effect on the reduction of viral replication. Log 10 pfuper lung was reduced from 6.9 in RSV-immunized control mice withoutIL-12 administration to 3.8 in immunized mice with IL-12 administration(FIG. 1). In contrast, IL-12 administration at the time of challenge didnot have significant effect on the viral replication (FIG. 1).

The effects of different delivery routes for IL-12 is described inExample 2. IL-12 was administered either intraperitoneally as describedin Example 1, or intramuscularly mixed with the RSV immunogen. Table 1shows the effect of either intraperitoneal or intramuscular delivery ofIL-12 on the reduction of viral replication. A single dosage of IL-12given simultaneously with immunogen had the same effect on the reductionof viral replication compared to the 5-dosage intraperitoneal regimen.IL-12 as a specific immunomodulator only worked in RSV immunized mice,having no effects on the unprimed mice (FIG. 1, Table 1). These datademonstrate that IL-12 exerts a potent adjuvant effect on theinactivated RSV immunogen.

The patterns of immunoglobulin isotypes produced in response toimmunization are indirect indicators of the types of cytokines producedin vivo. IgG2a is produced in mice as a consequence of Th1 cellactivation, whereas IL-4 promotes the production of IgG1 (Burstein, H.J. et al, J. Immunol. 147:2950 (1991); Finkelman, F. D., et al, AnnuRev. Immunol. 8:303 (1990); Morris, S. C., et al, J. Immunol. 152:1047(1994)). As described in Example 3, blinded assays of serum RSV-specificimmunoglobulin isotype titers in RSV-immunized mice receiving IL-12showed that IL-12 induced significantly more RSV-specific IgG2a antibodyand significantly less IgG1 antibody compared to immunized mice notgiven IL-12. The pattern of IgG2a and IgG1 RSV-specific antibodyresponse was similar whether IL-12 was given intramuscularly orintraperitoneally (Table 2).

The pattern of antibody isotype utilization induced by IL-12 suggeststhat in vivo IL-12 administration can promote the differentiation ofantigen-specific CD4+ Th1 cells and inhibit the development of Th2 cellsin response to the inactivated RSV intramuscular immunization. Thepattern of cytokine mRNA expression in lungs was directly examined asdescribed in Example 4. Lung tissues from immunized mice, with orwithout IL-12 treatment, were harvested at 4 days after live viruschallenge. The cytokine mRNAs for IFN-γ, IL-4, IL-6, IL-10, and IL-12,were measured by Northern blot analysis. There were no obviousdifferences in IL-6 and IL-10 mRNA levels among mice with differenttreatments. However, the lungs from mice treated with IL-12 at the timeof either immunization or challenge contained more IFN-γ relative toIL-4 compared to control mice that did not receive IL-12 (FIG. 2).Increased IL-12 MRNA expression occurred in the mice treated with IL-12at the time of immunization, while IL-12 administration at challenge didnot increase IL-12 mRNA expression (FIG. 2). These data suggest thatIL-12 enhanced Th1 cell differentiation and produced a shift from a Th2to a Th1-like response in mice immunized with the inactivated RSVimmunogen.

RSV-specific cytotoxic T lymphocyte (CTL) activity in lungs of immunizedmice was assessed to evaluate whether IL-12 administration enhancedcell-mediated immunity as a positive modulatory effector. As describedin Example 5, a direct CTL assay using lung lymphocytes was employedwhich does not include in vitro stimulation (Tang, Y.-W., et al, J.Clin. Invest. (1994)). There was no difference in CTL activity betweengroups that received or did not receive IL-12 treatment at the time ofimmunization. This result, which was repeated in two consecutiveexperiments, further suggests the Th1-like cytokine pattern was aproduct of CD4+ T cells. It is reasonable to expect that altering thedose of IL-12 would induce a greater CD8+ response which would alter theillness pattern.

As described in Example 5, even though IL-12 has a dramatic effect onthe reduction of RSV replication in lungs, there was not significantdifference in the clinical outcome, including weight loss and illnessscore between groups. The simple shift in the pattern of cytokineexpression was therefore not predictive of a change in illness. Thissuggests that the cell populations responsible for cytokine productioncan be key determinants of illness and not cytokines themselves. Forexample, mice treated with anti-IL-4 at the time of immunization alsohad increased IFN-γ expression in lungs at the time of challenge.However, the anti-IL-4 treatment resulted in diminished illness that wasassociated with increased CD8+ CTL activity (Tang, Y.-W., et al, J.Clin. Invest. (1994)). In the case of anti-IL-4 treated mice, it may bethat the IFN-γ was a product of CD8+ T cells, whereas in IL-12 treatedmice, CD4+ T cells are a more likely source.

Adjusting the dose of IL-12 can alter its properties. A recent study ofIL-12 on immune responses to lymphocytic choriomeningitis (LCMV)infection showed that low doses of IL-12 enhanced immunicity to LCMVinfection as demonstrated by increased splenic CD8+ T cell numbers anddecreased LCMV replication. However, high doses of IL-12, equivalent tothose used in the examples, impaired resistance against LCMV infectionas demonstrated by reduced virus-specific CTL activity and increasedviral replication (Orange, J. S. et al, J. Immunol. 152:1253 (1994)). Itmay therefore be possible to adjust the dose of IL-12 or its method ofdelivery to maintain the effect on viral inhibition, but to also impactillness.

The invention is further illustrated in the following examples.

EXEMPLIFICATION Example 1 Immunization of Mice with RSV and IL-12

Mice: Pathogen-free female BALB/c mice, 8 to 10 months old, werepurchased from Charles River Laboratories (Raleigh, N.C.) and cared foraccording to the “Guide for the Care and Use of Laboratory Animals” aspreviously described (Graham, B. S., et al, J. Med. Virol. 26:153(1988)).

RSV Immunogen and Virus: Preparation of the formalin-inactivatedalum-precipitated RSV and preparation of stock of the A2 strain of RSVhave been previously reported (Graham, B. S., et al, Immunol. 151:2032(1993)). Both the vaccine preparation and the challenge stock werederived from the A2 strain of RSV.

Murine Cytokine IL-12: Murine recombinant IL-12 was expressed fromcloned cDNAs (Schoenhaut, D. S., et al, J. Immunol. 148:3433 (1992)).The lot used in this paper was MRB021693-1.2 (Genetics Institute,Cambridge, Mass.) with a specific activity of 5.6×10⁶ units/mg asdetermined by PHA blast assay (Wolf, S. F., et al, J. Exp. Med. 146:3074(1991)). Concentrated aliquots of IL-12 were stored at −70° C. anddiluted in phosphate-buffered saline with 1% normal mouse serum (1%PBS).

Immunization: Mice were immunized with formalin-inactivatedalum-precipitated RSV containing 2.2×10⁶ pfu equivalents of virusantigen intramuscularly, and challenged with 10⁷ pfu of live RSVintranasally 4 weeks later as previously described (Graham, B. S., etal, Immunol. 151:2032 (1993)); Tang, Y.-W., et al, J. Clin. Invest.(1994)). IL-12 was administered intraperitoneally for 5 successive days,starting at one day before immunization at a dose of 1 μg/mouse. Controlmice received 1% phosphate buffered saline (PBS) on the same schedule.

The viral replication in lungs 4 days after challenge was assessed byplaque assay. Mouse serum samples were collected on the day of and twoweeks after live RSV challenge.

Plaque Assays and Neutralization Tests: Two-day old HEp-2 monolayers,80% confluent in Costar 12-well plates, were used for plaque assay andneutralization tests. The assays were performed as described previously(Graham, B. S., et al, J. Med. Virol. 26:153 (1988)).

IL-12 administered at the time of immunization has a marked effect onthe reduction of viral replication. Log 10 pfu per lung was reduced from6.9 in RSV-immunized control mice without IL-12 administration to 3.8 inimmunized mice with IL-12 administration. In contrast, IL-12administration at the time of challenge did not have significant effecton the viral replication. See FIG. 1. (Log 10 pfu/gram lung is shown asarithmetic means±S.D.; KV denotes killed virus)

Example 2 Effect of Delivery Route of IL-12 on its Adjuvant Ability

The effect of a different delivery route of the adjuvant IL-12 wasassessed. IL-12 was administered intraperitoneally as described inExample 1 to one group of mice. To another group of mice, IL-12 wasadministered intramuscularly mixed with the RSV antigen. The controlmice were either mock immunized or treated with IL-12 alone withoutantigen. Table 1 summarizes the results of the experiment which showthat a single dosage of IL-12 given simultaneously with immunogen hadthe same effect on the reduction of viral replication compared to the5-dosage intraperitoneal regimen. IL-12 as a specific immunomodulatoronly worked in RSV immunized mice, having no effects on the unprimedmice. See FIG. 1 and Table 1. These data demonstrate that IL-12 exerteda potent adjuvant effect on the inactivated RSV immunogen.

Example 3 Assay of Immunoglobulin Isotype Titers in RSV-immunized MiceReceiving IL-12

The patterns of immunoglobulin isotypes produced in RSV-immunized micereceiving IL-12 was examined. Mouse serum samples were collected on theday of and two weeks after live RSV challenge.

RSV-Specific Immunoglobulin Isotype ELISA: All serologic assays wereperformed by a person blinded to the experimental groups. BCH4 and BCcells were bound to the solid phase on Immulon II 96-well plates (NUNC,Denmark). Serial diluted mouse serum samples were added to each well.Plates were incubated, washed, and goat anti-murine IgG1 or IgG2aconjugated to alkaline phosphatase (Southern Biotechnology, Birmingham,Ala.) diluted 1:1000 was added, respectively. After another incubation,plates were washed and substrate was added for 30 minutes at roomtemperature and OD₄₀₅ was determined (Graham, B. S., et al, Immunol.151:2032 (1993); Tang, Y.-W., et al, J. Clin. Invest. (1994)). A serumdilution was considered positive if the mean optical density of two BCH4cell wells was greater than twice that of BC-coated wells and greaterthan 0.1.

The results demonstrate that IL-12 induced significantly moreRSV-specific IgG2a antibody and significantly less IgG1 antibodycompared to immunized mice not given IL-12. The pattern of IgG2a andIgG1 RSV-specific antibody response was similar whether IL-12 was givenintramuscularly or intraperitoneally. See Table 2.

Example 4 Pattern of Cytokine mRNA Expression in Lungs from MiceImmunized with and without IL-12

Lung tissues from immunized mice, with and without IL-12 treatment, wereharvested at 4 days after live virus challenge. The cytokine mRNAs forIFN-γ, IL-4, IL-6, IL-10 and IL-12 were measured by Northern blotanalysis.

mRNA Extraction, Northern Blotting, and Cytokine Detection: The totalRNA from whole lungs was extracted and polyA RNA isolated,electrophoretically separated and transferred to membrane as previouslydescribed (Graham, B. S., et al, Immunol. 151:2032 (1993); Tang, Y.-W.,et al, J. Clin. Invest. (1994)). Hybridization with ³²P oligonucleotideprobes were performed as previously described (Graham, B. S., et al,Immunol. 151:2032 (1993)). After washing, membranes were exposed toKodak X-omat film at −70° C. Laser densitometry was performed with anLKB UltroScan XL using GelScan XL software (Pharmacia Fine Chemicals,Piscataway, N.J.). Oligonucleotide probes for murine IL-4, IL-10, IFN-γ,IL-6 were purchased from R &: D Systems (Minneapolis, Minnesota) orClontech Laboratory Inc. (Palo Alto, Calif.). A cocktail ofoligonucleotides designed for detecting IL-12 was based on the murineIL-12 sequence spanning predicted splice sites based on those identifiedin the human IL-12 sequence. (Schoenhaut, D. S., et al, J. Immunol.148:3433 (1992)). From 5′ to 3′:

p-40: p-40: TGAGGACACATCTTGCTTTGCTGCGAGCTG, (SEQ. ID. NO:1)TCCCGCCTTTGCATTGGACTTCGGTGATG, (SEQ. ID. NO:2) andCAACGTTGCATCCTAGGATCGGACCCTGCA; (SEQ. ID. NO:3) p-35:GCCAGGCAACTCTCGTTCTTGTGTAGTTCC, (SEQ. ID. NO:4) andGCGTTGATGGCCTGGAACTCTGTCTGGTAC. (SEQ. ID. NO:5)

Cytokine mRNA Northern blots (2 samples per group) with averagedα-tubulin-normalized densities are shown in FIG. 2. There were noobvious differences in IL-6 and IL-10 RNA levels among mice withdifferent treatments. However, the lungs from mice treated with IL-12 atthe time of either immunization or challenge contained more IFN-γrelative to IL-4 compared to control mice that did not receive IL-12(FIG. 2). An increased IL-12 mRNA expression occurred in the micetreated with IL-12 at the time of immunization, while IL-12administration at challenge did not increase IL-12 mRNA expression (FIG.2). These data suggested that IL-12 enhanced Th1 cell differentiationand produced a shift from a Th2 to a Th1-like response in mice immunizedwith the inactivated RSV immunogen.

Example 5 RSV-specific Cytotoxic T Lymphocyte (CTL) Activity in Lungs ofImmunized Mice

RSV-specific CTL activity in lungs of immunized mice was assessed toevaluate whether IL-12 administration enhanced cell-mediated immunity asa positive modulatory effector. A direct CTL assay using lunglymphocytes was employed which does not include in vitro stimulation(Tang, Y.-W., et al, J. Clin. Invest. (1994)).

Cytotoxicity T Cell Assays: Whole lung lymphocytes were isolated byFicoll-Hypaque (1.09 specific gravity) cushion centrifugation. BCH4 andBC target cells labeled with ⁵¹Cr (Dupont-New England Nuclear, Boston,Mass.) were incubated with effector cells for 4 hours at 37° C. in96-well microtiter plates as described (Tang, Y.-W., et al, J. Clin.Invest. (1994)). The spontaneous and total release were obtained bytreating the target cells with 10% RPMI and 5% Triton X-100 detergent,respectively. Each point was the mean from three replicate wells. Thespecific release of ⁵¹Cr from target cells was defined as 100× (samplecpm−background cpm)/(total cpm−background cpm).

There was no difference in CTL activity between groups that received ordid not receive IL-12 treatment at the time of immunization. This resultwas repeated in two consecutive experiments and further suggests theTh1-like cytokine pattern was a product of CD4+ T cells. Even thoughIL-12 has a dramatic effect on the reduction of RSV replication inlungs, there was no significant difference in the clinical outcome,including weight loss and illness score between groups. Illnessassessment, including weight loss and clinical scores were performed aspreviously described (Tang, Y.-W., et al, J. Clin. Invest. (1994)). Thesimple shift in the pattern of cytokine expression was therefore notpredictive of a change in illness. This suggests that the cellpopulation responsible for cytokine production may be a key determinantof illness and not cytokines themselves. For example, mice treated withanti-IL-4 at the time of immunization also had increased IFN-γexpression in lungs at the time of challenge. However, the anti-IL-4treatment resulted in diminished illness that was associated withincreased CD8+ CTL activity (Tang, Y.-W., et al, J. Clin. Invest.(1994)). In the case of anti-IL-4 treated mice, it may be that the IFN-γwas a product of CD8+ T cells, whereas in IL-12 treated mice, CD4+ Tcells were a more likely source. TABLE 1 IL-12 Administered EitherIntraperitoneally or Intramuscularly Reduces Virus Replication in Lungsand Noses IL-12 Log 10 pfu/gram Log 10 pfu/ Group N Immunogen RouteLung* Nose* 1 5 KV — 6.56 ± 0.44 3.73 ± 0.15 2 5 KV IP 4.75 ± 0.25† 2.61± 0.23‡ 3 5 KV IM 4.44 ± 0.55† 2.80 ± 0.34‡ 4 5 Mock — 6.55 ± 0.32 3.73± 0.29 5 5 Mock IP 6.51 ± 0.34 3.21 ± 0.27*Mean ± S.D.†p < 0.001 compared to Log 10 pfu/gram lung in group 1‡p < 0.001 compared to log 10 pfu/nose in group 1KV killed virusIP intraperitonealIM intramuscularly

TABLE 2 IL-12 Administered Alters RSV-Specific Serum ImmunoglobulinIsotype Titers at and Two Weeks After RSV Challenge IL-12 At Challenge 2Weeks After Challenge Group Immunogen Route IgG1 IgG2a IgG1 IgG2a 1 KV —<80 (0/5)* <80 (0/5) 640.0 ± 2.2 (4/4) 269.1 ± 4.2 (2.4) 2 KV IP <80(0/5) 557.2 ± 6.4† (3/5) 121.3 ± 1.9‡ (2/5) 640.0 ± 6.7‡‡ (3/5) 3 KV IM<80 (0/5) 367.6 ± 4.1 (3/5) <80 (0/5) 905.1 ± 4.2‡‡ (3.4) 4 Mock — <80(0/5) <80 (0/5) <80 (0/5) 113.1 ± 2.1 (1/4) 5 Mock IP <80 (0/5) <80(0/5) <80 (0/5) 139.3 ± 2.5 (2/5)*Number converted/number tested†Geometric mean titer ± S.D. Negative samples were assigned a titervalue of 80 for statistical calculations.‡p < 0.01 compared to IgG1 2 weeks after challenge in group 1‡‡p > 0.5 compared to IgG2a 2 weeks after challenge in group 1KV killed virusIP intraperitonealIM intramuscular

1-18. (canceled)
 19. A composition for reducing replication of a virusof the Paramyxoviridae family in a host, comprising a mixture of anantigen of the virus and an effective adjuvant amount of interleukin-12.20. A composition of claim 19 wherein the virus of the Paramyxoviridaefamily is selected from the group consisting of: parainfluenza virus 1,parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4,mumps virus, measles virus, respiratory syncytial virus, caninedistemper virus, bovine respiratory syncytial virus, Newcastle diseasevirus and rhinderpest virus.
 21. A composition of claim 19 wherein theantigen is selected from the group consisting of: the fusionglycoprotein, the hemagglutinin-neuraminidase, the G glycoprotein, andan inactivated Paramyxoviridae.
 22. A composition of claim 19 whereinthe composition optionally contains one or more of other adjuvants,pharmaceutically acceptable surfactants, excipients, carriers, diluents,vehicles, sweetening agents, flavoring agents, and coloring agents. 23.A composition of claim 19 wherein the antigen is present as apolynucleotide and is expressed in vivo.
 24. A composition of claim 19wherein the virus of the Paramyxoviridae family is respiratory syncytialvirus.
 25. A composition of claim 24 wherein the antigen is selectedfrom the group consisting of: the fusion glycoprotein, the Gglycoprotein, and an inactivated respiratory syncytial virus.
 26. Acomposition of claim 24 wherein the composition optionally contains oneor more of other adjuvants, pharmaceutically acceptable surfactants,excipients, carriers, diluents, vehicles, sweetening agents, flavoringagents, and coloring agents.
 27. A composition of claim 24 wherein theantigen is present as a polynucleotide and is expressed in vivo.